Process for controlling hydrogen ion concentration of butyl alcohol fermentation mashes



Patented Dec. 8, 1936 PRUMESS IFQR 'IUUNTROLLING RUGEN KQNCONCENTRA'JELJIUN @F Ell MCQ- HWL FJERMENTA'JTMDN MASHIES David A. lLeggand ugh R. Stiles, Terre Manta, llnd., assignors to @oercial Solvents@orporation, Terre lilaute, End... a corporation at Maryland Nolllrawing. Application February 12, 19%,,

Serial No. No.89?

5 Claims.

certain physical forms of basic carbonates, hereinbelow described.

It is known that in a number of fermentation processes, particularlythose in whichdntermediate products and/or end products of an acidicnature are produced, the metabolic functions of the organisms arefavored by a hydrogen ion con: centration in the mash lower than thatsecured by the normal activity of the microorganisms themselves. It hasbeen the custom to maintain the desired hydrogen ion concentration bymeans of a water-insoluble basic material such as calcium carbonate,barium carbonate, etc. The materials used for this purpose in commercialscale fermentations have been ground limestone or calcite, usually ofabout 200 mesh size, or coarser. Such materials, when present in excessin the mash, serve to neutralize acidity when formed and thus maintainthe hydrogen ion concentration within the necessary limits forsatisfactory fermentation. In certain fermentations, for example thoseby means of organisms of the type Clostridium propyl butylicum(described in copending application Serial No. 650,036 by Muller andLegg, filed January 3, 1933) or by, organisms of thetype Clostridzuminverto-acetobutylicum (described in 'co-pending application Serial No.675,458 by Legg and Stiles, filed June 12, 1933), the optimum hydrogenion concentration is more critical than in the usual types offermentations having acidic intermediate products, and the yield will befound to drop markedly if the hydrogen ion concentration varies onlyslightly from the optimum. In such cases it is seen that it is esandwhich are so designated in the appended claims, comprise any bacteriahaving the following primary characteristics:

I. Morphological A. Rod-shaped B. Spore-formingClostridia and PlectridaC. Practically indistinguishable from members of the Clostridiumbutyricum group II. Biochemical A. Carbohydrate fermentatiorr 1.Inability to produce appreciable yields I of butyl and isopropylalcohols from starch as the only source of carbohydrate 2. Inability toproduce appreciable yields 1% of butyl and isopropyl alcohols fromsucrose as the only source of carbohydrate 3. Inability to consistentlyproduce yields greater than 20% calculated on the 20 weight of the sugarfrom uninverted molasses 4. Ability to produce high yields of butyl andisopropyl alcohols from glucose or 25 inverted molasses B. Nitrogenmetabolism 1. Ability to produce high yields of butyl and isopropylalcohols in sugar media containing ammonia as the principal source ofnitrogen 2. Ability to utilize-degraded protein (including ammonia) assole nitrogen source 3. Inability to utilize undegraded protein as solesource of nitrogen 4. Inability to liquefy gelatin or to produce morethan very slight proteolysis of milk: C. Oxygen requirements 1.Anaerobic D. Temperature range for solvent production 1. From 25 C. to36 0., preferably 29 C. to 31 C. E. Hydrogen ion concentration forsolvent production 1. Final pH of 5.0-6.5, preferably 5.8-6.1 45

ill

The bacteria designated herein as Clostridium inverto-acetobutylicumcomprise any bacteria having the following primary characteristics:

I. Morphological A. Rod-shaped B. Spore-forming-Clostridia and PlectridaC. Practically indistinguishable from members of the Clostridiumbutyricum group II. Biochemical A. Carbohydrate fermentation 1.Inability to produce appreciable yields of butyl alcohol and acetonefrom starch as the only source of carbohydrate 2. Inability to produceappreciable yields of butyl alcohol and acetone from sucrose as the onlysource of carbohydrate 3. Inability to consistently produce yieldsgreater than 20% calculated on the weight of the sugar from uninvertedmolasses 4. Ability to produce high yields of butyl alcohol and acetonefrom glucose or inverted molasses B. Nitrogen metabolism 1. Ability toproduce high yields of butyl alcohol and acetone in sugar mediacontaining ammonia as the principal source of nitrogen 2. Ability toutilize degraded protein (including ammonia) as sole nitrogen source 3.Inability to utilize undegraded protein as sole source of nitrogen 4.Inability to liquefy. gelatin or to produce more than very slightproteolysis of milk C. Oxygen requirements 1. Anaerobic D. Temperaturerange for solvent production 1. From 25 C. to 36 0., preferably 29 C.

to 31 C. E. Hydrogen ion concentration for solvent production 1. FinalpH of 5.0-6.5, preferably 5.7-6.1

In the past it has been found to be extremely difiicult to duplicate, ona commercial scale, laboratory fermentations, particularly those bymeans of organisms of this latter type. It has been found that althoughall of the fermentation conditions prevailing in the laboratoryexperiment are duplicated, and an excess of ground calcite orcommercially available insoluble neutralizing agent is present at alltimes during fermentation, the hydrogen ion concentration in thecommercial scale fermentation is usually higher than that secured in thelaboratory, with a resulting decrease in yield. It has now beendiscovered that the cause for this unexpected change in hydrogen ionconcentration under apparently the same conditions is due to thedifferences in the shape and size of the vessels employed. In theordinary laboratory fermentation vessel, i. e. the small Erlenmeyerflask (flat-bottomed conical) of the order of 500 c. c. to 1000 c. c.capacity, the ratio of height of mash and volume of mash to surface areaof settled carbonate is quite low and the absolute height of the mashover the layer of settled carbonate is likewise quite low. In the usualtype of commercial scale fermentation vessel, on the other hand, thesefactors are greatly increased. The resulting effect may be readilydemonstrated by the following simple laboratory experiment.

the flask was covered with liquid. The yields secured are shown in thetable below.

Table I Yield of total solvents Tilted flasks Control flasks Grams perPercent on Grams per Percent on liter wt. of sugar liter wt. of sugar Itmay be seen from the above results that a marked decrease in yield wasobtained in the tilted flasks, i. e., when the ratio of height of mashand volume of mash to surface area of settled carbonate wassubstantially increased. It should be noted that the yields in thecontrol flasks do not represent the optimum yields obtainable with thisculture since the sugar con centration employed was above the optimum.However, the comparative results obtained in this experiment arerepresentative of the effect of changing the shape (and the associatedratios referred to) of the fermentation vessel.

It is thus seen that the eflicacy of one specific agent, viz., 200 meshcalcite in maintaining the desired hydrogen ion concentration depends to.a large extent upon the size and shape of the vessel employed. It isbelieved that a body of mash in any particular vessel has a space factordependent upon the ratio of height of mash' where H=height of mesh incm., V=volume of mash in cm. and A=surface area of the layer of settledcarbonate in cmfi.

It has been found, for example. that in a series of fermentatlons ofmolasses mash by means of Clostridium mopyl butylicum, carried out inwidely different shaped and sized vessels, and again employing 200 meshcalcite as the hydrogen ion regulator, a yield of over 30% was obtainedin only two cases. These were laboratory fermentations in an Erlenmeyerflask and a beaker, both having space factors" below 3. In the othervessels, having space factors ranging from 3.19 to 3.73, the yieldsranged from 29% to as low as 23.8%. These results are shown in the tablebelow.

Table II Volume Height Area Of (HI/)1 Yield Type of vessel of of +(H) lon wt.

mash mash bonate A f 7 layer 0 sugar 10- Cm (I'm 4 liter Erlenmeyerflask. 3.0 18. 0 302. 0 2. 21 30. 1 2 liter beaken. l. 2 6.8 132. 5 2.08 31. 5 1 liter roundbottom flask. 0. 7 6. 9 34. 3 3. 29 29. 0 2 iterroundbottomflask; 1.7 10.2 61.7 3. 47 27.6 550 gallon fetmenter 11050171 9450 3. 72 26. 2 10,000 gallon lei-mentor--- 18900 185 46800 3.1928. 9 10,000 gallon lermenter..- 23280 232 48500 3.49 25.4 10,000 gallonlei-mentor- 28040 262 50200 3. 73 23.8

It may be seen from the above table' that good yields were obtained onlyin the laboratory fermentations in the beaker and the Erlenmeyer flask.However, it is apparent that even in the case of the 4 liter Erlenmeyerflask the space factor tended to decrease the yields somewhat. As hasbeen previously mentioned, in the case of the usual small Erlenmeyerflasks (i. e. of the order of 5000. c. to 1000 c. c.) nosuch effect canusually be observed. It may also be seen from the table that fair yieldswere obtained in the 1 liter round-bottom flask (space factor=3.29) andin the half-filled fermenter (space' factor: 3.19), and poor yields wereobtained in the remaining cases (space factors=3.47 and upwards). If theexpression for the space factor given above is assumed to beapproximately correct, it may be seen that factors above 3.0 result indecreased yields, and factors above 3.3 result in poor yields.

In spite of this discovery of the cause of the decreased yields in thecommercial scale fermentations, it was found that none of the expectedremedies were successful. An increase in the excess of calcite presentwas found to have no effect, or, if increased unduly, was found to havea detrimental effect. Agitation of the mash to secure better contact ofthe calcite generally resulted in decreased yields of the desiredproducts. The continuous introduction of suitable neutraliz ing agentsfailed because of the mechanical difficulties involved. Numerous otherattempts to remedy this situation by buffering the mash or by othersimilarmeans for hydrogen ion control have lilrewise failed.

The surprising discovery has now been made that the use of calciumcarbonate or other insoluble non-tonic neutralizing agent in asubstantially more finely divided state than that previously employedwill result in full yields duplieating those obtained in laboratoryexperiments in small Erlenmeyer flasks. This is accomplished without thenecessity of agitating the mash or adding any further neutralizingagents during the fermentation. Materials suitable for this purpose areprecipitated barium, iron, or calcium carbonates, or ground naturalmaterials of a substantially equally fine state of division. Oftheseinaterials, the calcium carbonates are preferred, owing to theircheapness and availability. A number of varieties of precipitatedcalcium carbonate are available for use in the manufacture of toothpaste, cosmetics, and the like, or for use as pure laboratory reagents.The naturally occurring calcium carbonates, e. g. chalk, limestone,etc...

may be subjected to continued grinding until a sufliciently finelydivided product is obtained, or may be passed in suspension through acolloid mill to yield the desired product. All of these materials aresuitable for use in the present invention but due to their cost it ispreferred to utilize a freshly precipitated calcium carbonate which mayadvantageously be precipitated from a lime suspension by means offermentation gases containing a substantial amount of carbon dioxide.

It is to be understood, of course, that this invention is not to belimited to any particular neutralizing agent or to any method ofpreparing the same. Any calcium carbonateor other insoluble non-toxicneutralizing agent which presents an available surface during thefermentation substantially greater than that presented by the standard200 mesh calcite will be found to give improved results. The availablesurface presented will, of course, depend upon the buoyancy of theparticles or aggregates of the material during fermentation. This, ofcourse, will in turn depend upon the size, surface structure andapparent density of the particles. Although the absolute test of thesuitability of a given material should be-made under fermentationconditions so that the gas evolution may play its part in maintainingthe carbonate in a state of suspension, an approximate comparativedetermination suitable for most purposes may be made by a simplesettling test from an aqueous suspension. Such a test is illustratedbelow.

Suspensions of 1.5 grams of each of a number of calcium carbonates in500 c. c. of distilled water were placed in 500 c. c. graduatedcylinders. The contents of the graduates were shaken by inverting twelvetimes and were then allowed to settle. At intervals the apparent volumeof solids on the bottom and the degree of turbidity were noted. Theresults obtained for four types of calcium carbonate are recorded in thetable below.

Table III Apparent volume of 1m solids on bottom in c. c. Turbxdlts Typeoicarbonate 11 2 1 4 1 5e r1 2 1 41 56 mm. min. IIllI]. mm. mm. mm. min.min.

Wet OaOOa p r e c i p itated from lime by fermentetion gas 6.0 7.5 8.59.0 M M M-L L C o s m e t i c grade precipitated OaCO 2.5 3.0 4.0 4.5 DD M-D M Commercial g r o u n d chalk 3.5 3.7 4.0 4.0 M 1V1. L L 200 m es h g r o u n d I limestone.-. 5.0 5.0 5.5 5.5 M-L L L L-C D=denseM=medium L light O=clear title other precipitated carbonates,nnit{natalng the presence of a considerable amount of very smallparticles. It is seen therefore that in the case of the precipitatedcalcium carbonates either the apparent volume of solids was greater orthe turbidity of the supernatant liquid was greater than in the case ofthe ground calcites. This test therefore should be sufficient in mostcases to enable one skilled in the art to choose a suitable type ofcalcium .carbonate.

Although the mass and apparent density of the particles would be a moreaccurate criterion because of differences in surface structure andporosity of the particles, a sufficiently accurate determination formost purposes may be made by ascertaining the average size of theparticles or aggregates. Such measurements will be found in most casesto follow closely the results obtained in the-settling test as will beshown by the results below, which were obtained with the same calciumcarbonates reported in Table III above.

The above results weresecured by examining a drop of a distilled watersuspension containing three grams per liter of calcium carbonate. The.drop was mounted in a quartz chamber and examined in a dark field with amicroscope fitted with a calibrated drum micrometer.

It may be seen from the above results that a marked difference existsbetween the precipitated calcium carbonates and the commercial grades ofground calcite. Thus, it is believed to be evident that by means of adetermination of both particle size and settling time one skilled in theart may readily choose a neutralizing agent sufficiently finely dividedto remain in suspension to the desired extent during the fermentationand thus to maintain the optimum hydrogen ion concentration. However, itmay be said that in general a neutralizing agent which presents anavailable surface during fermentation substantially greater than thatpresented by a slight excess of 200 mesh calcite, or which has a rate ofsettling from an aqueous suspension substantially lower than that of 200mesh calcite, or which has an average particle size substantially lessthan that of 200 mesh calcite will be satisfactory when employingvessels having a ratio of height and/or volume of mash to surface areaof settled neutralizing agent exceeding that obtained in smallErlenmeyer flasks. By

the term substantially in this connection is meant sufficiently to showa measurable increase in yield in parallel fermentations in which 200mesh calcite is compared with the finer material in vessels ofunfavorable space factor.

The beneficial effect upon the hydrogen ion concentration, throughoutthe fermentation, which is secured by the use of finely dividedneutralizing agents is illustrated in Table V below. The resultsreported in this table represent observations of the pH at differentstages of the fermentation of an inverted molasses mash by means ofClostridium propyl butylz'cum, following the standard procedure setforth in co-pending application Serial No. 650,036.

Table V Hydrogen ion concentration as H Yield percent Type of carbonatebased 20 40 4o 63 Inmal hrs. hrs. hrs. hrs. Sugar Wet CaCOs precipitatedfrom limeby fermentation gas..." 6.31 5.59 5.70 5.98 5.85 29.6 Cosmeticgrade precipitatcdCaCO: 5.66 5.61 5.63 5.80 5.54 20.4 Commercial groundchalk 5.49 5.44 5.53 5.50 5.32 28.5 200 mesh ground lime stone 5.49 5.395.43 5.51 5.26 28.9

The above results were obtained in fermentations of three liters of mashin four liter Erlenmeyer flasks. The effect of the type of neutralizingagent on the yield would probably be less in the case of smallErlenmeyer flasks (i. e., of the order of 500 c. c. to 1000 c. c.) andwould undoubtedly be more pronounced in the case of larger vessels.

The improved yields to be obtained in carrying out this invention areillustrated in Table VI below. These results were obtained in thefermentation of an inverted molasses medium by means of C'Zostridiumprom/Z bwfiylicum. The medium was prepared by hydroiyzing molasses ofabout 20% sugar concentration by heating with sulphuric acid equivalentto 5% on the weight of the sugar for 40 minutes at 20 lbs. pressure. Atthe conclusion of this inversion 0.7% of ammonia on the weight of thesugar was added and sumcient lime introduced to neutralize the remainingfree acidity. An excess of freshly precipitated calcium carbonate (orground chalk in other cases) amounting to 6% on the weight of the sugarwas then introduced and the mash diluted to a sugar concentration ofabout 5% and sterilized for 30 minutes at 20 lbs. pressure. Thefermentations were carried out in the same fermenters (10,000 gal.capacity) which were used for the last three fermentations reported inTable II, above. In each case the mash was inoculated with about 3% ofan active culture of Clostridium prcpyl butylicum and incubated at 32 C.The table gives the initial and final hydrogen ion concentrations, aswell as the yields and solvent ratios obtained.

Table VI Solvent ratio Yield. 3 53 of Hydrogen ion regulator 33 percentB t l Ethyl and on Sugar u Acetone isopropyl alc. B103 6120 gals.--Ground chalk 5. 89 5. 23 25. 4 69. 0 16. 9 14. 1 6145 gals. CaCO;precipitated from 0.20 5. 85 31.0 69. 0 12. 1 l8. 9

lime by fermentation gas. 7510 $115.... Ground chalk 5. 94 5. 21 24. 070. 3 12. 3 17.4 8220 gals--. OaCO; precipitated from 5. 98 5. 71 31. 267. 2 4. 7 28. 1

lime by fermentation gas.

As may be seen from the above results, an increase in yield of.from 22%to 30% on the weight of the total solvents was obtained whenprecipitated carbonate rather than commercial ground chalk was employed.When it is considered that a single fermentation plant may producemillions of pounds of solvents in the course of a year the importance ofsuch an increase in yield may readily be seen.

It is recognized .that precipitated calcium carbonate has often beenutilized for neutralizing lib tilt

till) fermentation vessel.

lift

the usual laboratory fermentations in small Erlenmeyer flasks. This,however, has been due to its availability as a laboratory reagent andnot because of any'improved result obtained thereby. As has beenpreviously pointed out, in the case of laboratory fermentations inshallow vessels having a relatively large bottom surface, satisfactoryresults are obtained with commercial ground calcite, and in most casesno improvement may be noted when a more finely divided material isemployed. The present invention, therefore, is limited to fermentationsin which the ratio of height of mash and/or volume of mash to area ofsettled carbonate substantially exceeds that obtained in laboratoryfermentations in small Erlenmeyer flasks i. e., in vessels with a spacefactor of the order of 3.0 and upwards. By the term substantially inthis connection is meant sufficiently to show a measurable decrease inyield in parallel fermentations in which the vessel in question iscompared with a small Erlenmeyer flask utilizing 200 mesh calcite inboth cases.

It is thus seen that in carrying out our invention improved results maybe obtained in any of the common types of commercial scale fermenta,

tion vessels merely by insuring a substantially greater availablesurface area of neutralizing agent during the fermentation than thatsecured by the use of the commercial grades of ground calcite in thesame vessel. It should be noted that the term available surface ofneutralizing agent as used here signifies the total exposed surface ofthe particles, as opposed to the term "surface area of the layer ofsettled neutralizing agent as used elsewhere in this specification. Thelatter term signifies only the exposed surface area of the layer ratherthan the total exposed surface of the particles.

Although improved results may be secured in practically all cases merelyby utilizing a neutralizing agent of smaller average particle size thanthat of 200 mesh calcite, in order to secure optimum results in anyparticular case it may be desirable to use a specific form ofneutralizing agent. A large variety of types of neutralizing agents,especially calcium carbonates are readily available, ranging from 100mesh or coarser down to the finest precipitated materials. One skilledin the art will usually be able to choose the cor rect material for agiven fermentation -merely from an inspection of the size and shape ofthe it. rough but usually sufficient estimate may be made by correlatingthe space factor of the vessel with the degree of fineness of theneutralizing agent-the higher the space factor the finer theneutralizing agent to be employed and vice versa. However, a moreaccurate determination may very readily be made by carrying out a simplepreliminary experiment in the form of a series of fermentations such asthose reported in Table V above, using materials "iii of differentaverage particle size.

templated fermentation conditions and the correct choice of neutralizingagent is insured. It is thus seeh that in' its broadest aspects ourinvention relates to the use of a neutralizing agent of a degree offineness corresponding to the size and shape of the body of fermentingmash in which it is to be employed.

It is to be understood, of course,that this invention is not to belimited to any of the specific examples given above. Although theinvention, was illustrated in connection with fermentations by means ofClostridium mopyl butylz'cum, it is applicable to other ferm'entationshaving acidic intermediate and/or end products, as for example,fermentations by means of Clostridium inverto-acetobutylicum. In generalit may be said that the invention is applicable to all fermentations bymeans of organisms whose metabolic functions are favored by a hydrogenion concentration in the mash lower than that secured by the normalactivity of said microorganisms. Likewise, the invention is not to belimited to any of the particular types of neutralizing'agents mentioned.All insoluble nontoxic neutralizing agents having an available surfaceduring the fermentation substantially greater than that presented by 200mesh calcite are included within the scope of this inventionf Likewise,the amount of neutralizing'agent to be used in any particularfefinentation may vary, depending upon the type of products secured. Forexample, although 6% of calcium carbonate on the weight of the sugar wasspecified for the fermentation by Clostrz'dium prom Z butylicum,

in the art from the nature ofthe particular fermentation. Othermodifications or improvements may be combined with the presentinvention.. For example, if a very active fermenta tion is involved, itmay be desirable to accommodate forthis activity in the choice of theneutralizing agent in accordance with co-pending application Ser. No.710,898, filed February 12, 1934. In general it may be said that anymodifications or the use of any equivalents which would naturally occurto one skilled in the art are included within the scope of thisinvention.

The invention now having been described, what is claimed is: I

l. In the fermentation of a carbohydrate mash by means of butylalcohol-producing bacteria whose metabolic functions are favored by ahydrogen ion concentration lower than that secured by the normalactivity of the organisms in said mash, the mash being contained in avessel shaped to have a space factor greater than 8.0, where Hrepresents the height ofthe mash in centimeters, V represents the volumeof the mash in cubic centimeters, and A represents the area of the layerof settled neutralizing agent in square centimeters, the im provementwhich comprises effecting the fermentation in the presence of a finelydivided non-toxic insoluble basic neutralizing agent in a concentrationslightly in excess of that required to neutralize initial acidity, saidneutralizing The amount to be till agent presenting an available surfaceduring fermentation greater than that presented by an equal excessconcentration of mesh calcite in said mash.

2. In the fermentation of a carbohydrate mash by means of butylalcohol-producing bacteria whose'metabolic functions are favored by ahydrogen ion concentration lower than that secured by the normalactivity of the organisms in said mash, the mash being contained in avessel shaped to have a space factor gg ma greater than 3.0, where Hrepresents the height of the mash in centimeters, V represents thevolume of the mash in cubic centimeters, and A represents the area ofthe layer of settled neutralizing agent in square centimeters, theimprovement which comprises effecting the fermentation in the presenceof a finely divided non-toxic insoluble basic neutralizing agent in aconcentration slightly in excess of that required to neutralize initialacidity, said neutralizing agent having a rate of settling from'anaqueous suspension lower than that of 200 mesh calcite.

3. In the fermentation of a carbohydrate mash by means of butylalcohol-producing bacteria whose metabolic functions are favored by ahydrogen ion concentration lower than that secured by the normalactivity of the organisms in said mash, the mash being contained in avessel shaped to have a space factor greater than 3.0, where Hrepresents the height of the mash in centimeters, V represents thevolume of the mash in cubic centimeters, and A represents the area ofthe layer of settled neutralizing agent in square centimeters, theimprovement which comprises effecting the fermentation in-the presenceof a finely divided nontoxic insoluble basic neutralizing agent in aconcentration slightly in excess of that required to neutralize initialacidity, said neutralizing agent having the average size of itsparticles and aggregates less than that of 200 mesh calcite.

4. In the fermentation of a carbohydrate mash by means of bacteria ofthe type Clostridium prom l butylicum, the mash being contained in avessel shaped to have a space factor greater than 3.0, where Hrepresents the height of the mash in centimeters, V. represents thevolume of the mash in cubic centimeters, and A represents the area ofthe layer of settled neutralizing agent in square centimeters, theimprovement which comprises effecting the fermentation in the presenceof a finely divided nontoxic insoluble basic neutralizing agent in aconcentration slightly in excess of that required to neutralize initialacidity, said neutralizing agent presenting an available surface duringfermentation greater than that presented by an equal excessconcentration of 200 mesh calcite in said mash.

5. In the fermentation of a carbohydrate mash by means of bacteria ofthe type Clostridium invertoracetobutylicum, the mash being contained ina vessel shaped to have a space factor greater than 3.0, where Hrepresents the height of the mash in centimeters, V represents thevolume of the mash in cubic centimeters, and A represents the area ofthe layer of settled neutralizing agent in square centimeters, theimprovement which comprises effecting the fermentation in the presenceof a finely divided non-toxic insoluble basic neutralizing agent in aconcentration slightly in excess of that required to neutralize initialacidity, said neutralizing agent presenting an available surface duringfermentation greater than that presented by an equal excessconcentration of 200 mesh calcite in said mash.

DAVID A. LEGG. HUGH R. STJLES.

